Isolation and Identification of a Novel Circovirus Associated with Turbot (Scophthalmus Maximus) Acute Hemorrhage Disease

Abstract

Turbot (Scophthalmus maximus) is an important economic farming fish in China. However, since November 2019, an emerging disease outbroke in turbot farms, characterized by severe bleeding in the fish fins and named turbot acute hemorrhage disease (TAHD). The TAHD could lead to great mortality in one or two weeks, with accumulated mortality exceeding 90%. The TAHD spread rapidly across the major turbot farming regions in China and has resulted in significant economic losses. From the diseased fish, we discovered a novel circovirus by virome sequencing, named turbot circovirus (TCV). The particle of TCV is approximately 30 nm and located in the fish spleen and kidney. The genome of TCV is 1774 bp, with three open reading frames (ORFs) encoding the replication protein (Rep), capsid protein (Cap), and ORF3 of unknown function. Compared with the reported circovirus sequences, the similarities of the TCV Rep sequence were lower than 53%. Phylogenetic analysis of the Rep and Cap protein sequences revealed that TCV is a novel circovirus. The PCR detection method was established to detect the clinical samples of TAHD from 2019, and all the samples showed TCV positive. Various cell lines, including turbot kidney (TK), epithelioma papilloma cyprinid (EPC), chinook salmon embryo (CHSE), and spotted halibut kidney (SHK), were used for TCV isolation; however, no cytopathic effect (CPE) was observed. The diseased fish tissue homogenate filtrate was used for experimental infection, and all the infected turbot showed symptoms as natural infection, with increasing cap gene copies post infection. Based on these results, we supposed that the novel circovirus TCV was closely associated with TAHD. Our research indicated that the circovirus may be a great threat to fish, and we should pay more attention to the circovirus in aquaculture.

Keywords: Turbot, Turbot acute hemorrhage disease, Circovirus, Pathogen